Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Precise probes of type II interferon activity define the origin of interferon signatures in target tissues in rheumatic diseases.

doi: 10.1073/pnas.1209724109

Figure Lengend Snippet: Fig. 4. Distinct patterns of type I and type II IFN-induced proteins in SS and DM tissue biopsies. (A) Protein lysates made from control (CS, n = 6) and SS (SS, n = 8) MSG biopsies were probed for IFN-inducible protein expression by Western blotting. Markers of type I IFN (MDA5, IFIT3) and type II IFN (GBP1, GBP2) were analyzed. Vinculin is included as a loading control. (B) Protein lysates made from control (CM, n = 3) and DM (DM, n = 4) muscle biopsies were probed with markers of type I and type II IFN. Vinculin is included as a loading control. (C) IFN-induced protein expression from A and B was quantified by densitometry and normalized to the level of vinculin expres- sion in the respective sample. Vinculin-normalized expression values were median centered and subject to unsupervised hierarchical clustering to de- fine patterns of IFN-induced protein expression in individual patients.

Article Snippet: Briefly, after rehydration, antigen retrieval, and blocking, sections were incubated overnight at 4 °C with either anti-IFIT3 (10 μg/mL; Novus) or GBP2 antibodies (30 μg/mL; Novus Biologicals).

Techniques: Control, Expressing, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Precise probes of type II interferon activity define the origin of interferon signatures in target tissues in rheumatic diseases.

doi: 10.1073/pnas.1209724109

Figure Lengend Snippet: Fig. 5. Colocalization of markers of type I and type II IFN activity in SS salivary gland biopsies. Minor salivary gland biopsies from controls (n = 3) and patients with SS (n = 4) were stained with antibodies against the type I IFN-preferential protein IFIT3 (A–C) and the IFN-γ–specific protein GBP2 (D–F). Representative images from one control (A and D) and two SS (B, C, E, and F) biopsies are shown. Staining with isotype control antibodies was negative in all samples (shown in Fig. S5). The asterisks denote areas of inflammation, and the arrows designate salivary gland epithelial cells that express both IFIT3 and GBP2. (Scale bars, 50 μm.)

Article Snippet: Briefly, after rehydration, antigen retrieval, and blocking, sections were incubated overnight at 4 °C with either anti-IFIT3 (10 μg/mL; Novus) or GBP2 antibodies (30 μg/mL; Novus Biologicals).

Techniques: Activity Assay, Staining, Control

Journal:

Article Title: Bacterial Peptide Recognition and Immune Activation Facilitated by Human Peptide Transporter PEPT2

doi: 10.1165/rcmb.2008-0059OC

Figure Lengend Snippet: Lung epithelial expression of nucleotide-binding oligomerization domain (NOD)-related factors. Total RNA and protein were obtained from primary human upper airway epithelial cells (hLECs) after overnight culture with or without 250 units/ml of IFN-γ, or 100 ng/ml of TNF-α. (A) NOD1/CARD4 and (B) NOD2/CARD15 mRNA was quantified by real-time PCR and expressed as relative copy number (RCN). GAPDH and CAP were used as internal controls to standardize and quantify mRNA expression. (C) Whole cell lysates were immunoprecipitated with affinity-purified anti-Nod1/Card4 antibody (IMGENEX), and then immunoblotted with an anti-Nod1/Card4 antibody (provided by Dr. Dana Philpott). Pre-immune rabbit serum was used as a negative control. (D) Western analysis of RIP2. Samples were loaded with 40 μg/well protein from the same lysate, and then the membrane was blotted with an anti-RIP2 antibody. β-Actin served as control to determine equal loading. (E) An immunoblot demonstrating constitutive levels of NOD1 and RIP2 proteins in both BEAS-2B and hLEC lung epithelial cells; 80 μg/well protein was loaded for each sample.

Article Snippet: GAPDH and CAP were used as internal controls to standardize and quantify mRNA expression. ( C ) Whole cell lysates were immunoprecipitated with affinity-purified anti-Nod1/Card4 antibody (IMGENEX), and then immunoblotted with an anti-Nod1/Card4 antibody (provided by Dr. Dana Philpott).

Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Affinity Purification, Negative Control, Western Blot, Membrane

Journal:

Article Title: Bacterial Peptide Recognition and Immune Activation Facilitated by Human Peptide Transporter PEPT2

doi: 10.1165/rcmb.2008-0059OC

Figure Lengend Snippet: γ-iE-DAP stimulates interaction between NOD1 and RIP2, leading to NF-κB activation. (A) Immunoprecipitation of human NOD1 was performed in BEAS-2B whole cell extracts after transfection with flag-tagged NOD1 and treatment with γ-iE-DAP (5 μg/ml). (A) Samples were resolved and then immunoblotted with an anti-RIP2 antibody. Western analysis was also performed with a monoclonal anti-flag antibody (Sigma) and β-actin (lower panels), demonstrating equal amounts of NOD1, specifically in the NOD1-transfected cultures (lanes 3–6) and equal loading in all samples. Negative controls included immunoprecipitation with a mouse IgG isotype control antibody (lane 1) or empty vector transfected cells (lane 2). (B) siRNA Suppression of NOD1 expression: since constitutive levels of NOD1 are barely detectable, we evaluated siRNA efficacy in BEAS-2B exposed to IFN-γ. Compared with the scrambled control siRNA, siRNA targeted to NOD1 resulted in a significant reduction in IFN-γ–induced protein levels. (C) Next we evaluated the ability of α-iE-DAP and γ-iE-DAP to induce NF-κB–mediated transcription in baseline, siControl, and siNOD1-treated cultures. First, BEAS-2B cultures were transfected with siRNAs, followed by transfection with an NF-κB/luciferase promotor plasmid. At 4 hours after transfection, cells were administered γ-iE-DAP or α-iE-DAP (5 μg/ml each) for 2 hours. Lysates were than analyzed for firefly luciferase activity and normalized to renilla expression. We observed that only γ-iE-DAP treatment resulted in luciferase expression, and this was significantly decreased in cultures treated with siRNA against NOD1 (data are expressed as mean values ± σ of n = 3; P ≤ 0.01). (D) To further validate these findings, BEAS-2B cultures were transiently transfected with siRNA against NOD1 or a scrambled control and then exposed to γ-iE-DAP or α-iE-DAP. Muropeptides were removed after 4 hours and then cells were cultured in normal medium for an additional 20 hours. As shown, only γ-iE-DAP treatment resulted in a significant increase in IL-6 release that was inhibited back down to baseline levels in cells receiving siRNA directed against NOD1 (data are expressed as mean values ± σ of n = 3; P ≤ 0.05).

Article Snippet: GAPDH and CAP were used as internal controls to standardize and quantify mRNA expression. ( C ) Whole cell lysates were immunoprecipitated with affinity-purified anti-Nod1/Card4 antibody (IMGENEX), and then immunoblotted with an anti-Nod1/Card4 antibody (provided by Dr. Dana Philpott).

Techniques: Activation Assay, Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Cell Culture

Journal: Cancer gene therapy

Article Title: PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

doi: 10.1038/s41417-024-00820-5

Figure Lengend Snippet: Fig. 2 The dynamic effect of differentiation markers followed by PHF10 in transiently transfected cells. A Parietal cell marker ATP4B. B Main cell marker Pepsinogen I. C Gastric neuroendocrine cell marker GAST. D Gastric mucus cell marker TFF1. E GC stem cell marker CD44. F Gastric progenitor cell marker SOX9. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756).

Techniques: Transfection, Marker

Journal: Cancer gene therapy

Article Title: PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

doi: 10.1038/s41417-024-00820-5

Figure Lengend Snippet: Fig. 3 PHF10 inhibits subcellular morphology and enhances sphere formation ability in GC cells. A SGC7901 cells exhibited numerous microvilli on the cell surface and pits on the nuclear membrane (×15,000). B SGC7901 cells manifested a significant presence of rough endoplasmic reticulum in the cytoplasm (white arrow), limited mitochondrial cristae (black arrow) (×60,000). C SGC7901-shPHF10 cells exhibited a decrease in microvilli and pits on the nuclear membrane (×10,000). D SGC7901-shPHF10 cells presented a notable quantity of lysosomes with high electron density (white arrow) (×60,000). E The cytoplasm of SGC7901-shPHF10 cell exhibited an increase in mitochondrial cristae (white arrow) (×60,000). F Cells with PHF10 knockdown displayed alterations in the Golgi complex (white arrow) (×30,000). G Cytoplasmic vacuolization was observed in SGC7901-shPHF10 cells (×30,000). H Apoptotic cells were identified in SGC7901- shPHF10 cells (×15,000). I–K The sphere formation capacity of SGC-7901, MKN28 and GES-1 cells manipulated by PHF10 was evaluated by measuring the number and diameter of primary and secondary spheres separately. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756).

Techniques: Membrane, Knockdown

Journal: Cancer gene therapy

Article Title: PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

doi: 10.1038/s41417-024-00820-5

Figure Lengend Snippet: Fig. 5 PHF10 serves as a member of SWI/SNF complex in GC cells. A, B The nuclear co-localization of PHF10 and BRG1 was observed in SGC7901 cells (A) and MKN28 cells (B). C–F Co-IP assays were performed using antibodies against PHF10 (C), BRG1 (D), BAF155 (E) and SNF5 (F) in SGC7901-shPHF10 and control cells. G–J Co-IP experiments for PHF10 (G), BRG1 (H), BAF155 (I) and SNF5 (J) were conducted in MKN28- PHF10 and control cells.

Article Snippet: The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Cancer gene therapy

Article Title: PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

doi: 10.1038/s41417-024-00820-5

Figure Lengend Snippet: Fig. 6 PHF10 directly targets DUSP5 through SWI/SNF complex. A–C Detection of E2F1, PHF10 and DUSP5 expression in SGC7901 cells by qRT-PCR. D–F The mRNA levels of E2F1, PHF10 and DUSP5 in MKN28 cells were also assessed. G, H WB analysis was performed to determine the levels of DUSP5, ERK1/2 and pERK1/2 levels in SGC7901 cells. I The levels of E2F1, PHF10, DUSP5, ERK1/2 and pERK1/2 levels in MKN28 cells were evaluated. J ChIP-qPCR was carried out using antibodies against PHF10, BRG1, BAF155 and SNF5 in SGC7901 cells, with IgG antibodies serving as a negative control. The purified DNA after precipitation was subsequently amplified using the same set of primers for PCR analysis. K The promoter region of DUSP5 was amplified in MKN28-PHF10 cells using antibodies against PHF10, BRG1, BAF155 and SNF5. L A dual luciferase reporter assay was conducted in both MKN28-PHF10-Vector and MKN28-PHF10 cells.

Article Snippet: The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756).

Techniques: Expressing, Quantitative RT-PCR, ChIP-qPCR, Negative Control, Luciferase, Reporter Assay, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

doi: 10.1038/s41417-024-00820-5

Figure Lengend Snippet: Fig. 7 PHF10 mediates differentiation disorders in GC through E2F1-PHF10-DUSP5-pERK1/2. A The relationship between E2F1 or PHF10 and DUSP5, was examined in 30 pairs of GC tissues through qRT-PCR analysis. B IHC staining was performed for E2F1, PHF10 and DUSP5 in two patient samples. Patient 1 exhibited poorly differentiated and diffuse GC, while patient 2 had well differentiated and intestinal GC. C WB analysis was conducted to measure the levels of E2F1, PHF10, DUSP5 and pERK1/2 in the aforementioned patient samples. D The expression levels of gastric epithelium differentiation markers (ATP4B, PG I, GAST, TFF1, CD44 and SOX9) were detected by qRT-PCR in SGC7901 cells. E The mRNA levels of gastric epithelium differentiation markers were assessed in MKN28 cells. F, G Sphere formation was quantified in SGC7901 cells (F) and MKN28 cells (G). H A schematic illustrating the mechanism of PHF10-mediated dysdifferentiation in GC cells.

Article Snippet: The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756).

Techniques: Quantitative RT-PCR, Immunohistochemistry, Expressing

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: ( A ) Binding kinetics of twofold dilutions of 3D11 Fab to PbCSP. Representative sensorgrams are shown in black and 2:1 model best fits in red. Data are representative of three independent measurements. ( B ) Isothermal titration calorimetry (ITC) analysis of 3D11 Fab binding to PfCSP at 37°C. Above, raw data of PbCSP (0.005 mM) in the sample cell titrated with 3D11 Fab (0.4 mM). Below, plot and trendline of heat of injectant corresponding to the raw data. K D and N values resulting from three independent experiments are indicated. Standard error values are reported as standard error of the mean (SEM). ( C ) Results from size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) for the 3D11 Fab-PbCSP complex. A representative measurement of the molar mass of the 3D11 Fab-PbCSP complex is shown as the red line. Mean molar mass and standard deviation are as indicated. ( D ) SDS-PAGE analysis of resulting Peaks 1 and 2 from SEC-MALS. Each peak was sampled in reducing and non-reducing conditions as indicated by + and -, respectively.

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques: Binding Assay, Isothermal Titration Calorimetry, Size-exclusion Chromatography, Multi-Angle Light Scattering, Standard Deviation, SDS Page

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: ( A ) 3D11 Fab binding to peptides representative of the PbCSP αTSR domain (residue 263–318; red) and the full C-terminal domain (residues 202–318; PbC-CSP; blue). PbCSP (residue 24–318) was used as a positive control (black). ( B ) Composite omit map electron density contoured at 1.0 sigma (blue mesh) around PbCSP peptides PAPP, NAND, NPND and Mixed in complex with the 3D11 Fab. ( C ) Slight differences in H-bonding at the N- and C-terminal ends of the PbCSP repeat peptides when bound to the 3D11 Fab in the crystal structures. PbCSP peptides are colored as in . Antibody residues partaking in H-bonds are colored orange. mAb 3D11 HCDR2 is colored in black and KCDR2 in white.

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques: Binding Assay, Residue, Positive Control

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: ( A ) Affinities of 3D11 Fab for PAPP, NAND, NPND, and Mixed peptides as measured by ITC. Symbols represent independent measurements. Mean K D values are shown above the corresponding bar. Error bars represent SEM. Peptide sequences are as indicated to the right of the plot, with variable residues underlined and shaded residues indicating those resolved in the corresponding X-ray crystal structures. ( B ) The 3D11 Fab binds the PAPP (pink), NAND (purple), NPND (blue) and Mixed (red) peptides in nearly identical conformations. mAb 3D11 CDRs are indicated. ( C ) Overview and side view of the NAND peptide (purple) in the binding groove of the 3D11 Fab shown as surface representation (H-chain shown in black and K-chain shown in gray). ( D ) Van der Waals interactions formed by side chain atoms of both Ala and Pro residues are indicated by orange dashed lines, and those unique to Pro6 and Pro10 are indicated by green dashed lines.

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques: Binding Assay

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: X-ray crystallography data collection and refinement statistics. Despite binding in nearly identical conformations, differences exist in the molecular details of 3D11 Fab binding to each peptide that provide key insights into mAb 3D11 recognition of PbCSP. Our crystal structures revealed that more van der Waals contacts were formed by a Pro residue in the PPPP and NPND motifs compared to an Ala at the same position in the PAPP and NAND motifs ( Figure 3D ). Consequently, the epitopes of the NAND, NPND and Mixed peptides had a slightly greater buried surface area (BSA; 753, 762, and 765 Å 2 , respectively) than the PAPP peptide (743 Å 2 ), which only consists of Ala-containing motifs ( Supplementary file 2 ). In particular, Pro10 of the PPPP motif found in the NAND and NPND peptides forms more van der Waals interactions with antibody residues H.Asn33 and H.Tyr52 compared to Ala10 of the PAPP motif present in PAPP and Mixed peptides. Similarly, Pro6 of the NPND motif in the NPND and Mixed peptides makes additional interactions with antibody residue K.Leu50 that are not present for Ala6 of the NAND motif within the PAPP and NAND peptides ( Supplementary file 2 ). These differences in interactions observed at the atomic level directly relate to the binding affinities measured by ITC, where the PbCSP peptides that bury more surface area in the 3D11 paratope have the highest binding affinities ( Figure 3A ).

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques: Structural Proteomics, Binding Assay, Residue

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: Interactions formed between PbCSP peptide residues and aromatic side chains of the 3D11 Fab HCDR (black) and KCDR (white). PbCSP peptides are colored as in .

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques:

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: ( A ) The cryoEM map of the 3D11 Fab-PbCSP complex reveals high-resolution information for seven predominant 3D11 Fabs. Regions corresponding to Fabs are colored from pink to gray. ( B ) CryoEM map of the 3D11 Fab-PbCSP complex is shown as a transparent light gray surface with the PbCSP region highlighted in black. ( C ) The PbCSP model built into the cryoEM map is shown in dark gray as sticks and aligned to the schematic representation of the PbCSP protein sequence.

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques: Sequencing

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: ( A ) Left panel – a representative cryoEM micrograph used to calculate the 3D11 Fab-PbCSP cryoEM map. Right panel – a cryoEM micrograph of the 3D11 Fab-PbCSP complex from a 200 kV screening microscope with individual particles highlighted with white circles. Scale bars: 50 nm. ( B ) Selected 2D class averages of the 3D11 Fab-PbCSP complex. ( C ) Particle orientation distribution plot. ( D ) Fourier shell correlation curve from the final 3D non-uniform refinement of the 3D11 Fab-PbCSP complex in cryoSPARC v2. ( E ) Local resolution (Å) plotted on the surface of the cryoEM map. ( F ) Low-pass filtered (20 Å) cryoEM map of the 3D11 Fab-PbCSP complex with observable 3D11 Fabs numbered. ( G ) CryoEM map of PbCSP (gray mesh) with the model shown as sticks (black carbons).

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques: Microscopy

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: ( A ) Color representation of the backbone RMSD of the 3D11 Fab variable region and PbCSP core epitope between the 3D11 Fab-PbCSP cryoEM structure and the 3D11 Fab-NPND peptide crystal structure. ( B ) Color representation of the all-atom RMSD of the 3D11 Fab residues engaging in Fab-Fab contacts between the 3D11 Fab-PbCSP cryoEM structure and the 3D11 Fab-NPND peptide crystal structure. RMSD values were calculated using PyMOL and plotted by color on the secondary structure of the 3D11 Fab-NPND peptide crystal structure.

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques:

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: ( A and B ) Close-up views of adjacent 3D11 Fabs from the cryoEM structure in complex with PbCSP (black). 3D11 Fabs bound to PbCSP form homotypic contacts with each adjacent Fab through two interfaces; one consisting of CDRs from the heavy and light chains of Fabs A and B (interface 1, A ), and the second mediated by residues in FR3 of Fab A HC and FR3 of Fab C LC (interface 2, B ). Variable domains of Fabs are shown in white. HCDR1, −2,–3, and KCDR1, −2 and −3 are colored yellow, orange, red, green, blue and purple, respectively. Residues forming Fab-Fab contacts are labeled with the position of the Fab in the cryoEM model (A, B or C) indicated in subscript. mAb 3D11 affinity-matured residues that engage in Fab-Fab contacts, but do not directly interact with PbCSP are highlighted in yellow with red font. Black dashed lines denote H-bonds. ( C ) Sequence alignment of mAb 3D11 with its inferred germline precursor. INT1 and INT2 refer to the two interfaces shown in ( A ) and ( B ). Green highlight: germline-encoded residues involved in homotypic interactions; Red: affinity-matured residues involved in homotypic interactions; Yellow highlight: affinity-matured residues involved in homotypic interactions that do not directly interact with PbCSP. ( D ) Binding affinity of WT 3D11 and H-58/73 germline-reverted mutant (Mut) Fabs to NPNDx1 (gray bars) and NPNDx2 (white bars) peptides as measured by ITC. Symbols represent independent measurements. Mean K D values resulting from at least two independent experiments are shown. Error bars represent standard error of the mean. An unpaired one-tailed t-test was performed using GraphPad Prism 8 to evaluate statistical significance: *p<0.05.

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques: Labeling, Sequencing, Binding Assay, Mutagenesis, One-tailed Test

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: ( A ) Main interaction interfaces between adjacent 3D11 Fabs in the 3D11 Fab-PbCSP cryoEM structure. ( B ) Table of contacts between 3D11 Fabs. HB: hydrogen bond (3.8 Å cut-off).

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques:

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: ( A ) SEC chromatogram of 3D11 IgG-PbCSP complexes. ( B ) Representative negative-stain (NS) micrographs of particles present in selected SEC fractions: 1 – soluble aggregates, 2 - 3D11 IgG-PbCSP complexes, 3 – unbound 3D11 IgGs. Scale bars: 100 nm. ( C ) Comparison of NS 2D class averages of the 3D11 IgG-PbCSP complex (upper panels) to 2D class averages of the 3D11 Fab-PbCSP complex (lower panels).

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques: Staining, Comparison

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet: CryoEM maps of the 3D11 Fab-PbCSP ( A and E ) and 311 Fab-PfCSP ( C and G ) complexes are shown as a transparent light gray surface with the CSP density highlighted in black for PbCSP and in blue for PfCSP. The CSP models built into the cryoEM maps are shown as gray for PbCSP; ( B and F ) or blue for PfCSP; ( D ) and H ) sticks and aligned to the schematic representations of their respective protein sequences.

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques:

Journal: eLife

Article Title: Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

doi: 10.7554/eLife.59018

Figure Lengend Snippet:

Article Snippet: Cell line ( Mus musculus ) , 3D11 hybridoma cell line , , BEI Resources #MRA-100; RRID: AB_2650479 , .

Techniques: Recombinant, Plasmid Preparation, Mutagenesis, Expressing, Transfection, Purification, Derivative Assay, Software

Journal: Nature

Article Title: Sterile protection against human malaria by chemoattenuated PfSPZ vaccine

doi: 10.1038/nature21060

Figure Lengend Snippet: Antibodies were assessed in sera taken before any immunizations (pre-immunization), two weeks following last immunization (post-immunization) and one day before CHMI (pre-CHMI). a–d, Antibodies were assessed to PfCSP by ELISA (a); air-dried PfSPZ by automated immunofluorescence assay (b); live PfSPZ by inhibition of sporozoite invasion (c); and 7,455 Pf peptides on a proteome array (d). a, PfCSP ELISA results are reported as net optical density (OD) 1.0; reciprocal serum dilution at which the optical density was 1.0 in post-immunization or pre-CHMI sera minus the OD 1.0 in pre-immunization sera. All negative net values were assigned a value of 1. Values above the dashed line are considered positive. b, Automated immunofluorescence assay (aIFA) results are reported as arbitrary fluorescent units (AFU) 2 × 105; reciprocal serum dilution at which the AFU were 2 × 105 in post-immunization and pre-CHMI sera. c, Inhibition of sporozoite invasion values are reported as the reciprocal dilution of pre-immunization, post-immunization and pre-CHMI sera that inhibited by 75% the numbers of PfSPZ invading as compared to in negative controls without serum. d, The 22 proteins on the proteome array recognized by post-immunization sera from at least five volunteers from group III (highest dose) are delineated. The list is derived from bipartite graph analysis following normalization and background correction using values from sera taken before injection of PfSPZ Challenge in vaccinees and controls, and after injection of normal saline in controls. The threshold of positivity for the array studies was more conservative compared to the ELISA analyses; for example, for PfCSP, 5 out of 9 array-positive compared to 9 out of 9 ELISA-positive. In a–c, protected individuals are represented in yellow and unprotected ones in grey, and box plots display median (middle line), 25th (lower hinge) and 75th (upper hinge) quartile. Whiskers extend to values within 1.5× the inter-quartile ranges of the lower and upper hinges, respectively.

Article Snippet: PfSPZ outside the hepatocytes were stained with an anti-PfCSP mAb (2A10, 6.86 μg ml −1 ) (Protein Potential LLC, with permission from New York University School of Medicine) conjugated with Alexa Fluor 633 (far-red) (1 μg ml −1 ; custom-conjugated at GenScript).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Inhibition, Derivative Assay, Injection, Saline

Journal: Nature

Article Title: Sterile protection against human malaria by chemoattenuated PfSPZ vaccine

doi: 10.1038/nature21060

Figure Lengend Snippet: Antibodies to PfCSP by ELISA were assessed in sera taken before any immunizations (pre-immunization), two weeks following last immunization (post-immunization) and 10 weeks after last immunization, which was one day before CHMI (pre-CHMI). PfCSP ELISA results are reported as net OD 1.0; the reciprocal serum dilution at which the optical density was 1.0 in post-immunization or pre-CHMI sera minus the OD 1.0 in pre-immunization sera. All values met criteria for positivity. Protected volunteers are represented by yellow circles and unprotected volunteers by grey circles.

Article Snippet: PfSPZ outside the hepatocytes were stained with an anti-PfCSP mAb (2A10, 6.86 μg ml −1 ) (Protein Potential LLC, with permission from New York University School of Medicine) conjugated with Alexa Fluor 633 (far-red) (1 μg ml −1 ; custom-conjugated at GenScript).

Techniques: Enzyme-linked Immunosorbent Assay